With CellViewer project we have been able to develop targeting strategies to Halo tag gene loci such as Oct4, Nanog, and β-catenin. In addition, we were able to develop efficient probes to efficiently label DNA, proteins and RNAs for SR imaging. As a result, we were able to visualize the DNA of gene loci and the corresponding mRNAs and protein products in living cells at nanoscale resolution. Further, we characterized the differences in RNA distribution after a treatment with a cell cycle inhibitor. We performed experiments to characterize protein kinetics in ESCs cultured in self-renewal condition and upon their differentiation
WP2 is focused on the visualisation of the dynamics of DNA, mRNA and proteins upon different perturbations. We imaged the spatial organization of DNA compaction in cells perturbed with Actinomycin D and Trichostatin A. Furthermore, we developed particle-tracking approaches and analysis algorithms to follow the dynamic of transcription factors and histone proteins in living mESCs cultured in pluripotency or differentiation conditions.
One of the major successes of CellViewer has been the successful building of a SR microscope prototype, which we expect to commercialise by the end of 2021. During 2020, the prototype will be beta-tested with high-throughput experiments for further improvement. This microscope contains an integrated software with a use-friendly interface to make it easier to use also for the non -expert users.
We have also been developing an integrated SR imaging analysis platform to analyse the high-throughput data obtained with our SR microscope. Indeed, we have developed new methods to extract information on the spatial organisation and architecture of proteins and DNAs within the nucleus of the cells. These imaging analysis methods will be used to extract info from the high-throughput data that will be collected with our prototype.
